• CNBr-activated sepharose 4B;
  • Protein Nα-acetylation;
  • Single-charge ion inclusion;
  • Technology


Protein N-terminal acetylation (Nα-acetylation) is among the most common modifications in eukaryotes. We previously described a simple method to enrich Nα-modified peptides using CNBr-activated Sepharose resin. A limitation of this method is that an optimal ratio of sample to resin had to be determined prior to the analysis since Lys-containing Nα-modified peptides may be lost. To address this problem, we hereby present an optimized method by the introduction of double incubation at pH 6.0. We demonstrate with the optimized method that the Nα-modified peptides can be enriched regardless of whether ε-NH2 is present or not, and the sample to resin ratio optimization is no longer necessary. Another improvement was accomplished by the inclusion of the singly charged precursor for MS/MS fragmentation to alleviate the shortcoming of the reduced charge state of Nα-modified peptides. We employed a duplicate experiment using 80 μg samples each and identified 922 IPI annotated and 103 IPI unannotated acetylated N-termini from 989 proteins, so far the largest acetylated N-termini data set acquired from a tryptic digest. Furthermore, the reproducibility of the Nα-acetyl proteome approach was evaluated and its complementarity to the regular proteome approach was analyzed. The unexpected coupling of CNBr-activated Sepharose to His-containing peptides via the imidazole group was discovered.